The long-term objective of this proposal is to increase our knowledge of the cellular mechanisms of insulin action. When insulin binds to its receptor at the cell surface signals are transmitted to the metabolic enzymes and glucose transporters regulated by insulin through signal transduction cascades. However, the molecular details of these pathways remain incompletely understood. Inhibition or disruption of these pathways leads to insulin resistance, and in susceptible individuals the development of diabetes. The focus of this proposal is the characterization of a unique insulin-stimulated signaling pathway that leads to tyrosine phosphorylation of caveolins-1 and -2, structural components of specialized cell surface domains termed caveolae. Tyrosine phosphorylation of the caveolins is both insulin-specific and cell type dependent, occurring only in adipocytes, suggesting that caveolin phosphorylation has an important function in these cells. Tyrosine phosphorylation promotes protein/protein interactions via SH2 domains, and the activation of downstream signaling cascades. Using a novel yeast-based phosphotyrosine dependent protein interaction screen, two proteins that interact with caveolin-1 in a phosphorylation-dependent manner were identified: TNFalpha receptor associated factor 2 (TRAF2) and C-terminal Src kinase (Csk). These findings indicate two important roles for caveolae in adipocytes: (1) modulation of TNFalpha signaling and (2) regulation of the actin cytoskeleton through Csk-mediated phosphorylation of the Src family kinases. These interactions implicate caveolae in TNFalpha-induced insulin resistance and in the insulin-induced actin rearrangements required for the stimulation of glucose transport in adipocytes. The specific aims of this proposal are to: 1. Determine the signaling pathways that lead to caveolin phosphorylation on both tyrosine and serine. 2. Determine the signaling pathways that lie downstream of caveolin phosphorylation (specifically TNF induced insulin resistance and regulation of the actin cytoskeleton and GluT4 translocation). 3. Examine the link between caveolin phosphorylation and metabolic regulation in vivo through the creation of fat-specific caveolin-1 and caveolin-1/Y14F knock-in mice.